Elemental Composition of Serpentine Plants Depends on Habitat Affinity and Organ Type Kyle S

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J. Plant Nutr. Soil Sci. 2014, 177, 851–859 DOI: 10.1002/jpln.201300485 851 Elemental composition of serpentine plants depends on habitat affinity and organ type Kyle S. DeHart1, George A. Meindl1, Daniel J. Bain2, and Tia-Lynn Ashman1* 1 Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA 2 Department of Geology and Planetary Science, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA

Abstract Serpentine soils represent a stressful growing environment for most plants due to a number of edaphic factors, including low concentrations of plant nutrients and high concentrations of heavy metals. Plants in these environments range from weakly resistant to strictly endemic, yet it re- mains unclear whether serpentine habitat affinity affects plant chemistry, including elemental distribution among various organs. We address this knowledge gap using three confamilial pairs of endemic and non-endemic plants. First, we determined total and phytoavailable soil concentrations of four nutrients (Ca, Mg, P, K) and three heavy metals (Co, Cr, Ni) across 11 serpentine study sites. Next, we determined the concentrations of these elements in leaves, flowers, and seeds in plants of each species growing on serpentine soil. Soils at the study sites were charac- teristically high in concentrations of Mg, Ni, Co and Cr, and low in K, P and Ca relative to non-serpentine soils. Habitat affinity was critical in determining the organ concentrations of Ca, Mg, K, and Co, although concentration often varied by organ type. Relative to non-endemics, endemics had higher concentrations of Mg and K across all organ types, whereas Ca concentrations were higher for non-endemics in the leaves but equal for the two reproductive organs. While no difference was observed in Ni or Cr concentrations, endemics contained 56% less Co than non-endemics across all organ types. These results suggest that serpentine endemics are more effec- tive at acquiring potentially limiting nutrients compared to non-endemic species, but both endemic and non-endemic plants exclude most phytotoxic heavy metals. Therefore, growth on serpentine requires common physiological responses of all plants, though high variation in uptake of some key nutrients and exclusion of some metals may reflect differential adaptation to serpentines by non-endemic and endemic plants.

Key words: endemic / heavy metals / plant chemistry / non-endemic / soil chemistry

Accepted September 01, 2014

1 Introduction populations in these areas experience strong selective pres- sures to survive and reproduce on (or off) serpentine, result- Serpentine soils are globally distributed, occurring on every ing in a mix of species that are either strictly adapted (i.e., en- continent and in virtually every type of climate (Brooks, 1987). demic), indifferent (i.e., with the ability to live both on and off), These soils are derived from Fe- and Mg-rich ultramafic rocks or intolerant (i.e., cannot survive on serpentine) to serpentine and are naturally enriched in heavy metals but low in plant nu- soils (Brady et al., 2005; Safford et al., 2005; Moore et al., trients (Alexander et al., 2007). Thus, serpentine-derived soils 2013). Plant adaptations to serpentine soils can include great- represent a nutritionally stressful environment for most plants er resistance to elevated concentrations of heavy metals because of: (1) low availability of Ca relative to Mg, (2) defi- (e.g., Ni) and Mg through exclusion or reduction in their trans- ciency of essential nutrients (e.g., N, P, K), and (3) high levels location (O’Dell et al., 2006; Palm et al., 2012). Other species of potentially phytotoxic heavy metals (e.g., Co, Cr, and Ni; may accumulate and sequester metals in a nontoxic form as Brady et al., 2005; Kazakou et al., 2008). As a consequence, a means to minimize the negative impact on plant function plants growing on serpentine soil must acquire scarce nu- and fitness (Kazakou et al., 2010). For example, Nagy and trients, while limiting excessive uptake of potentially harmful Proctor (1997a) found that plants from serpentine resistant trace elements (Brooks, 1987). populations of the species Cerastium fontanum, Festuca ru- bra, and Cochlearia pyrenaica had lower Ni concentrations in Across a landscape, serpentine soils are often embedded their tissues than did conspecifics from non-serpentine popu- within non-serpentine habitats, and as such they often rep- lations when grown in high Ni solutions. In addition, serpen- resent ‘islands’ that host novel plant species relative to the ex- tine endemics (e.g., Helianthus exilis, Mimulus nudatus, tensive matrix of non-serpentine soil and its plant commun- Streptanthus glandulosus ssp. pulchellus) can be more re- ities (Brady et al., 2005; Moore et al., 2013). As a result, plant sistant of depleted Ca and high Mg levels than congeneric

* Correspondence: Dr. T.-L. Ashman; e-mail: [email protected]

ª 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.plant-soil.com 852 DeHart, Meindl, Bain, Ashman J. Plant Nutr. Soil Sci. 2014, 177, 851–859 non-endemic serpentine species (e.g., Helianthus annuus, pected to lead to less than optimal nutrient acquisition for Mimulus guttatus) and non-serpentine species (Madhok, non-endemic serpentine species when found on serpentine 1965; Madhok and Walker, 1969; MacNair and Gardner, than in related endemics (Sambatti and Rice, 2006). 1998). For example, biomass yield of the common sunflower, Helianthus annuus, is greatly reduced with increasing soil Beyond species differences in nutrient acquisition, plants ex- Mg, whereas the serpentine endemic sunflower, H. bolanderi hibit organ-specific (e.g., leaves, flowers, seeds) patterns of ssp. exilis, is able to exclude Mg and thus increase yield de- elemental composition (Colangelo and Guerinot, 2006; Haw- spite levels of soil Mg that are toxic to H. annuus (Madhok kesford et al., 2012). This variation reflects the physiological and Walker, 1969). Moreover, some serpentine endemic spe- requirements of specific biological functions of the plant organ cies require higher concentrations of soil Mg compared to (Hawkesford et al., 2012). For example, Mg concentrations non-serpentine plants for optimal growth (Main, 1981). Thus, are usually highest in the leaves because it is a key compo- species or populations that vary in serpentine affinity often in- nent of chlorophyll molecules (Hawkesford et al., 2012). Like- teract differently with serpentine soils in terms of both resist- wise, most plant Ca is stored in the leaves where it serves ance and nutrient/element acquisition (Sambatti and Rice, structural roles in the cell wall and membranes among other 2007). functions (Karley and White, 2009). Furthermore, metals such as Ni are transported throughout the above-ground tissues of It is unclear whether serpentine endemic species are specifi- plants including the reproductive structures (i.e., flowers and cally adapted to the abiotic stresses of serpentine soils or seeds) and the vegetative tissue (i.e., leaves and shoots) rather are merely stress resistant species that rarely occur on where they may serve a defensive role (Colangelo and Gueri- non-serpentine soil. Two models have been proposed to de- not, 2006; Cheruiyot et al., 2013). High concentrations of po- scribe the occurrence of edaphic endemics: the ‘refuge’ mod- tentially toxic elements, such as metals, in plant reproductive el (Gankin and Major, 1964) and the ‘specialist’ model reduce fitness via negative impacts on pollen viability and pol- (Meyer, 1986; Palacio et al., 2007). Under the refuge model, linator visitation (Quinn et al., 2011; Meindl and Ashman, serpentine endemics are stress-resistant species that are not 2013). Due to the importance of organ-specific nutrient trans- strictly adapted to serpentine soils, but are able to resist ser- location and exclusion of metals, plant species entirely re- pentine soils and are eventually restricted there due to their stricted to serpentine soils may be better able to regulate ele- low competitive ability off of serpentine soils. Conversely, ment uptake and translocation relative to non-endemic ser- under the specialist model, serpentine endemics are specifi- pentine plants, though currently no study has addressed this cally well adapted to the abiotic stresses of serpentine soils, possibility. where they outcompete non-endemics. These models gener- ate specific, testable hypotheses relating to plant–soil interac- To address these knowledge gaps, we assessed organ-spe- tions. Specifically, under the specialist model, endemic plants cific concentrations of seven elements, four nutrients and should be more efficient at acquiring nutrients and/or limiting three heavy metals, from three confamilial endemic and non- uptake of potentially phytotoxic elements from the atypical endemic pairs of plants growing on serpentine soils. We ad- soils they inhabit relative to non-endemic species. Con- dress the following questions: (1) does serpentine habitat af- versely, under the refuge model, serpentine endemics and finity (i.e., endemic or non-endemic) lead to differences in the non-endemics are not expected to differ in uptake of either es- concentrations of plant elemental nutrients (i.e., Ca, Mg, P, K) sential or phytotoxic elements. However, few studies exist and/or heavy metals (i.e., Co, Cr, Ni) when grown on serpen- that have tested these models in natural populations by com- tine?; (2) are there differences in tissue elemental nutrient paring tissue chemistry of endemic and non-endemic species and/or heavy metal concentrations among organs (leaves, (Palacio et al., 2007). While a number of studies have flowers, seeds) and/or are the results dependent on habitat compared tissue chemistry between serpentine and non-ser- affinity? Specifically, we hypothesize (1) that endemics will in- pentine plant species (e.g., O’Dell et al. 2006), a paucity of corporate nutrients into their tissues (especially those impor- studies has compared tissue chemistry of endemic and non- tant for reproduction into reproductive organs) to a greater de- endemic serpentine species (Lee and Reeves, 1989; Nagy gree than non-endemic serpentine species, and (2) that en- and Proctor, 1997a; Burrell et al., 2012). demic species will exclude heavy metals from their tissues (especially those in reproductive organs) to a greater degree Understanding how endemics may differ from non-endemics than non-endemic serpentine species. has the potential to provide greater insight into adaptation to the serpentine environment. The close proximity of serpentine and non-serpentine soils increases the opportunity for gene 2 Material and methods flow between populations of a species that can grow on both soil types (Sambatti and Rice, 2006). In contrast, strong di- 2.1 Study site rectional selection for resistance to serpentine soil composition can produce serpentine endemics (Moore et al., 2013) The study site was located on the University of California’s that can no longer grow (or successfully compete) off serpen- Donald and Sylvia McLaughlin Natural Reserve in Lower tine, and ultimately become reproductively isolated from the Lake, California, USA (Napa, Lake, and Yolo Counties: parent species. Reproductive isolation between related ser- 38° 52¢ 26† N, 122° 25¢ 54† W). The climate in this region is pentine and non-serpentine taxa is often due to differences in Mediterranean, characterized by hot, dry summers from April phenology and pollination, which reduce gene flow (MacNair to October and cool, wet winters from November to March and Gardner, 1998; Gardner and MacNair, 2000). Thus, (Safford and Harrison, 2004; Drenovsky et al., 2013). The re- gene flow between individuals on and off serpentine is ex- serve contains a heterogeneous mix of serpentine and non-

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Table 1: Location (GPS coordinates) of eleven sampled serpentine 2005) in Napa and Lake Counties, California (Harrison et al., seeps at the McLaughlin Reserve. 2000; Beardsley and Olmstead, 2002; USDA, NRCS, 2013). The bushy perennial forb A. clevelandii, Cleveland’s milk- Serpentine seep Latitude (N) Longitude (W) vetch, and the annual forb T. obtusiflorum, clammy clover, are both native California flora (Safford et al., 2005), but A. cleve- RHA 38° 51¢ 32.75† 122° 24¢ 37.29† landii is endemic to serpentine soils (serpentine affinity = 6.1; RHB 38° 51¢ 31.29† 122° 24¢ 36.22† Safford et al., 2005) in the North and South Coast Ranges of northern California and is thus more restricted in its range 3RHB 38° 51¢ 29.24† 122° 24¢ 27.76† than T. obtusiflorum which can be found in the Peninsular, RHC 38° 51¢ 30.47† 122° 24¢ 34.61† Transverse, Sierra Nevada, California Coast Ranges, and RHD 38° 51¢ 36.81† 122° 24¢ 42.54† Cascade Range extending north into southwestern Oregon (serpentine affinity < 1; Safford et al., 2005; Harrison et al., TP8 38° 51¢ 40.14† 122° 25¢ 54.77† 2000; USDA, NRCS, 2013). Delphinium uliginosum, swamp 3TP8 38° 51¢ 32.09† 122° 25¢ 56.68† larkspur, and A. eximia, Van Houtte’s columbine, are both perennial forbs that are endemic to California (Safford et al., TPW 38° 51¢ 46.91† 122° 26¢ 53.51† 2005). Delphinium uliginosum occurs primarily on serpentine CS1 38° 51¢ 37.44† 122° 25¢ 15.29† soils (serpentine affinity = 5.7; Safford et al., 2005) in the In- ner North Coast Ranges, whereas A. eximia’s range is more BS 38° 51¢ 45.58† 122° 23¢ 57.91† widespread extending from southern to northern California PC 38° 52¢ 03.12† 122° 24¢ 07.63† (serpentine affinity = 4.2; Safford et al., 2005; USDA, NRCS, 2013). serpentine soils, leading to a marked differentiation in the physical and chemical features of the landscape (Safford and 2.3 Plant tissue and soil collections Harrison, 2004; Drenovsky et al., 2013), and supports diverse We collected leaves, whole flowers (i.e., perianth, stamens, serpentine plant communities (University of California-Davis pistils), and seeds (removed from fruits) from target species Natural Reserve System, 2000). Serpentine seeps are found and soil samples from eleven serpentine seeps (Table 1). For where springs flow from the permeable serpentine rock and each species and organ type we collected 21–30 samples in negotiate areas of sandy or gravelly soil that retain moisture total. Organs for individual species were collected across (Harrison et al., 2000; University of California-Davis Natural three to six seeps, with the exception of A. eximia, which only Reserve System, 2000). The combination of serpentine soil existed at two seeps. Collections were made every three me- with summer water results in a diverse habitat containing nu- ters along established transects within each seep, except for merous late-flowering serpentine non-endemic and endemic species with low abundances (A. clevelandii, T. obtusiflorum, plant species (Safford and Harrison, 2004; Drenovsky et al., and A. eximia), where collections were restricted by plant lo- 2013) of which six are studied here. cation. Newly developed leaves and flowers were collected during peak flowering. All organ collections were made at 2.2 Study species least 5 cm above the soil surface to avoid soil contamination. Six species constituting three confamilial species pairs (one While soil contamination of plant tissues is possible, particu- serpentine endemic and one serpentine non-endemic) used larly in regards to Cr (Mitchell, 1960; Cary and Kubota, 1990), in this study were: endemic Mimulus nudatus (historically in methods used to determine soil contamination of plant sam- Scrophulariaceae, but recently placed in Phrymaceae; ples are not without assumptions. For example, the Ti method Beardsley and Olmstead, 2002) with non-endemic Mimulus of estimating soil contamination for Cr (see Cary and Kubota, guttatus (Phrymaceae), endemic Astragalus clevelandii (Fa- 1990) assumes no Ti accumulation by plants; however, plants baceae) with non-endemic Trifolium obtusiflorum (Fabaceae), accumulate Ti in a species and organ-specific manner (Du- and endemic Delphinium uliginosum (Ranunculaceae) with mon and Ernst, 1988). Thus, we did not address soil contami- non-endemic Aquilegia eximia (Ranunculaceae). This paired nation of plant samples beyond thoroughly washing them. sampling strategy controls for effects of shared evolutionary Samples from each collection site were pooled by tissue type history (MacNair and Gardner, 1998) when testing for differ- (i.e., over multiple individuals) to achieve enough biomass for ences in element concentrations between serpentine endem- analysis. Seeds were collected from the same locations later ic and non-endemic plant species. Our definition of ‘endemic’ in the season. Plant material was stored in Ziploc bags with follows Safford et al., (2005) definition of ‘strict endemic’, at desiccant (Acros Organics, Morris Plain, NJ, USA) until proc- which ‡ 95% of species occurrences are on serpentine soil. essed. In addition, to characterize serpentine seep soil, espe- All other plants are considered ‘non-endemic’. cially with respect to the availability of the elements of inter- est, we collected soil at root depth (» 20–30 cm) from each Mimulus nudatus, the bare monkey-flower, and M. guttatus, plant sampling location (2–24 soil samples per seep). Soil the yellow monkey-flower, are both primarily outcrossing an- samples within seeps were pooled for analysis. nual forbs (Beardsley and Olmstead, 2002). Mimulus guttatus is native to western North America and has a widespread 2.4 Tissue and soil elemental analysis range from Mexico to Alaska (serpentine affinity < 1; Safford et al., 2005), whereas congener M. nudatus is restricted to Tissue samples were rinsed with deionized water and dried at the serpentine seeps (serpentine affinity = 5.6; Safford et al., 60°C for 24 h prior to elemental analysis. A 0.1g ground sam-

ª 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.plant-soil.com 854 DeHart, Meindl, Bain, Ashman J. Plant Nutr. Soil Sci. 2014, 177, 851–859 ple was microwave-digested in 4 mL of trace metal grade 3 Results HNO3 and brought to a final volume of 15 mL with MilliQ (Milli- pore, Bedford, MA, USA) H O. A 1 mL aliquot of diluted digest 2 3.1 Soil elemental composition was further diluted with 9 mL of 2% HNO3 solution and mixed with a small volume (80 mL) of known concentrations of three Soil from all the serpentine seeps had total concentrations of internal standards (beryllium, germanium, thallium). Concen- Mg ‡ 122 g kg–1, with phytoavailable concentrations ranging trations of seven elements (nutrients: Ca, Mg, P, K; metals: from 1.48 g kg–1 to 2.75 g kg–1 (Table 2). Total concentrations Co, Cr, Ni), known to differ between serpentine and non-ser- of K and P were both £ 500 mg kg–1, while phytoavailable pentine soils (Alexander et al., 1989; Kazakou et al., 2010), concentrations were £ 71.5 mg kg–1 for K and £ 3.71 mg kg–1 were determined via inductively coupled plasma mass spec- for P (Table 2). Total Ca concentrations were highly variable trometry (ICP-MS, Perkin/Elmer NEXION 300X) at the Univer- among the sites ranging from 3.7 to 13.2 g kg–1 with phyto- sity of Pittsburgh. Five standards with known element con- available concentrations ranging from 0.129 to 0.719 g kg–1 centrations were used to construct standard calibration (Table 2). Total heavy metal concentrations ranged from curves prior to each sample run on the ICP-MS. Blanks and 0.078 to 0.13 mg kg–1 for Co, from 0.63 to 1.64 g kg–1 for Cr, duplicate samples containing internal standards were ana- and from 1.43 to 2.67 g kg–1 for Ni (Table 2). Phytoavailable lyzed at regular intervals as a measure of quality control dur- concentrations for these metals were much lower compared ing sample processing on the ICP-MS. All processed dupli- to total concentrations (Co £ 0.801 mg kg–1;Cr£ 8.64 × 10–5 cate samples were within 10% of each other. gkg–1;Ni£ 1.46 × 10–2 gkg–1; Table 2). Magnesium was the most phytoavailable element in these soils, followed in de- Soil samples were air-dried and sieved (< 2mm) prior to creasing order by Ca, K, Ni, P, Co, and Cr. Soil pH was analysis. Soils were analyzed for pH (1 : 1 soil : dH2O), phyto- slightly alkaline for all studied sites ranging from 7.6 to 8.3 available and total element concentrations. Phytoavailable (Table 2). soil element concentrations were determined via ammonium acetate extraction (K, Ca, Mg) at pH 7.0 (Oze et al., 2003), di- ethylenetriaminepentaacetic (DTPA) acid extraction (Co, Cr, 3.2 Plant elemental composition Ni) at pH 7.23 (Lindsay and Norvell, 1978; Oze et al., 2003), Habitat affinity affected the tissue concentrations of all nu- and sodium bicarbonate (NaHCO ) extraction (P) at pH 8.5 3 trients (Ca, Mg, K) except P. The magnitude of the habitat af- (Olsen et al., 1954). Following the addition of extractant, sam- finity effect, however, was organ-specific and varied among ples were shaken (30 min NH OAc; 2 h DTPA; 30 min NaH- 4 the elements (Table 3). For two of the nutrients (Mg, K), en- CO ), centrifuged for 10 min at 3200 g, and the supernatant 3 demics had higher concentrations than non-endemics, but was decanted. Phytoavailable concentrations for all soil ele- the magnitude of the difference depended on organ type (Ta- ments were measured in the extractant using the ICP-MS at ble 3; Fig. 1). Endemics had » 28% more Mg in leaves and the University of Pittsburgh (accuracy and blank information flowers and 42% more Mg in their seeds than non-endemics are shown above). Total element concentrations for soil sam- (Fig. 1). Endemics had higher concentrations of K (16% ples were obtained via ICP-MS by ALS Minerals, Reno, NV, more) than non-endemics and this was consistent across all USA (Method ME-MS41) after aqua regia digestion. organ types (Fig. 1). The pattern among organs was variable for Ca. Calcium concentration was higher for non-endemics 2.5 Statistical analysis in the leaves (» 21% more), but more similar compared to endemics for the two reproductive organs [although endemics All statistical analyses were conducted in SAS (version 9.3; tended to have more Ca in their flowers (13%) and seeds SAS Institute Inc., Cary, NC, USA). All tissue element con- (16%); Fig. 1]. Overall, concentrations of Ca and Mg were centrations were non-normally distributed, and thus, were highest in leaves, while P was highest in seeds and K was log-10 transformed prior to analysis to improve normality. To highest in flowers (Fig.1). evaluate the effect of habitat affinity and organ type on element concentration, a mixed-model ANOVA was conducted Habitat affinity did not significantly influence the tissue con- (PROC MIXED) on each element separately (nutrients: Ca, centrations of Cr or Ni, but did influence the concentration of Mg, P, K; metals: Co, Cr, Ni). The model included the fixed ef- Co (Table 3). Endemics had 56% lower Co concentrations in fects of habitat affinity (endemic versus non-endemic), organ their tissues than non-endemics across all organ types (Ta- type (leaves, flowers, or seeds), and their interaction, and ran- ble 3; Fig. 2). Organs varied in their metal concentrations of dom factors of site, family, and species, in which species was Co and Cr, with leaves having highest concentrations (Co: nested within habitat affinity, site, and family. Significant inter- 8.7 × 10–4 – 2.2 × 10–4 gkg–1; Cr: 1.14 × 10–2 – 1.9 × 10–4 g actions were explored using SLICE, which partitions the inter- kg–1), followed by flowers (Co: 6.8 × 10–4 – 1.8 × 10–4 gkg–1; action of two factors so that each factor (here: habitat affinity) Cr: 6.35 × 10–3 – 1.7 × 10–4 gkg–1), and seeds (Co: 4.7 × 10–4 can be tested at different levels of the other factor (here: or- – 2.2 × 10–4 gkg–1; Cr: 4.26 × 10–3 – 1.7 × 10–2 gkg–1). There gan type; Schabenberger and Pierce, 2002; SAS Institute was no significant variation among organs in tissue Ni con- Inc., 2011). Least squares means (and 95% CI) (accounting centration (leaves: 1.19 × 10–2 – 2.6 × 10–4 gkg–1; flowers: for the random effect) were computed and subsequently 1.19 × 10–3 – 2.1 × 10–4 gkg–1; seeds: 1.15 × 10–2 – 2.2 × back-transformed for presentation purposes (Olsen, 2003). 10–4 gkg–1; Table 3).

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Table 2: Composition of soil (total and phytoavailable concentrations) from eleven serpentine seeps noted in Table 1 with respect to the seven focal elements. The geometric mean (– SE) composition (total and phytoavailable) across all seeps is also given. Units in g kg–1 (Ca, Mg, Fe, Cr, Ni) and mg kg–1 (P, K, Co).

Soil Seep Name metric RHA RHB 3RHB RHC RHD TP8 3TP8 TPW CS1 BS PC Mean

pHdH2 O 7.8 8.0 7.9 7.7 8.0 8.1 8.3 7.6 8.3 8.2 7.9 8.0

Total

Ca 6 7.1 13.2 6.8 9.3 3.7 4.6 4.1 3.9 7.1 6.6 6.6 – 0.8

Mg 132 137 135 122 137 152 174 141 146 153 142 143 – 4.1

P 260 160 220 240 310 220 380 290 260 300 240 262 – 17.4

K 500 200 200 300 300 500 300 400 300 300 400 336 – 31

Co 84.1 78.4 97.1 132 131 107 99.1 92.9 128 90.4 82.8 102 – 5.98

Cr 1.02 1.22 1.2 1.64 1.46 0.63 0.89 0.85 0.97 1.02 0.88 1.07 – 0.09

Ni 1.42 1.57 1.75 2.44 2.16 1.81 2.03 1.63 2.67 1.69 1.47 1.88 – 0.12

Phytoavailable

Caa 0.32 0.24 0.41 0.23 0.49 0.41 0.72 0.47 0.13 0.63 0.62 0.42 – 0.06

Mga 1.48 2.44 2.12 2.75 2.34 1.73 2.45 2.48 1.8 2.21 2.01 2.16 – 0.11

Pb 3.71 0.367 0.961 1.51 1.76 0.723 2.41 3.46 0.566 2.4 1.51 1.76 – 0.34

Ka 67 32 28.3 42.6 33.1 45.2 38.8 71.5 25.6 52.7 48.7 44.1 – 4.53

Coc 0.801 0.307 0.386 0.726 0.643 0.43 0.333 0.747 0.243 0.585 0.48 0.52 – 0.06

Crc 7.6 × 10–5 5.1 × 10–5 6.5 × 10–5 5.0 × 10–5 5.4 × 10–5 6.4 × 10–5 8.6 × 10–5 7.2 × 10–5 6.6 × 10–5 8.2 × 10–5 6.0 × 10–5 6.6 × 10–5 – 3.7 × 10–6

Nic 1.3 × 10–2 5.2 × 10–3 8.9 × 10–3 1.2 × 10–2 1.4 × 10–2 5.2 × 10–3 5.7 × 10–3 1.2 × 10–2 6.7 × 10–3 1.5 × 10–2 6.5 × 10–3 9.5 × 10–3 – 1.1 × 10–3 a extracted with NH4OAc at pH 7.0; b extracted with NaHCO3 at pH 8.5; cextracted with DTPA at pH 7.23.

14 25 –1 –1 12 Ca 20 Mg 10 8 15 6 10 4 5 2

0 Concentrationkg / g 0 Concentrationkg / g Leaves Flowers Seeds Leaves Flowers Seeds

6 1 – –1 5 P 20 K 4 15 3 10 2 1 5

0 kg Concentration / g 0 Concentrationkg / g Leaves Flowers Seeds Leaves Flowers Seeds Figure 1: Mean Ca, Mg, P, and K concentrations in leaves, flowers, and seeds as a function of species habitat affinity (i.e., endemic or non- endemic). Dark bars indicate endemics, while white bars indicate non-endemics. Error bars represent 95% confidence intervals.

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Table 3: (a) F values of fixed effects [habitat affinity (i.e., endemic or non-endemic) and organ type] from mixed-model ANOVAs on nutrient and heavy metal concentrations of tissues. (b) F values of significant interaction slices for the effects of habitat affinity and organ type (L = leaves, F = flowers, S = seeds). (c) Random effect covariance parameter estimates from mixed-model analyses of nutrient and metal concentrations. (Habitat Affinity = HA; Organ Type = OT).

Nutrients Metals

(a) Source of variation df Ca Mg P K Co Cr Ni

HA 1, 24 0.01 27.58**** 0.00 8.26** 8.62** 0.67 0.04

OT 2, 439 311.30**** 759.04**** 918.14**** 291.39**** 34.34**** 100.21**** 0

HA · OT 2, 439 9.81**** 5.33** 3.18* 1.82 1.24 0.44 0.34

(b) Slices by OT

Leaves: HA 1, 439 4.91* 14.10*** 0.22 ––––

Flower: HA 1, 439 0.65 17.82**** 2.05 ––––

Seeds: HA 1, 439 1.27 37.27**** 0.72 ––––

Species (HA Site Family) 24 0.008304 0.006114 0.002685 0.001023 0.04790 0.02358 0.05701

Site 10 0.002572 0 3.488 × 10–7 0 0 0.006686 0.003399

Family 2 0.01693 0.03356 0.001316 0.007004 0.06144 0.03499 0

* P < 0.05 ** P < 0.01 *** P < 0.001 **** P < 0.0001 Note: Random effects were included in model to account for their variation but were not tested for significance.

4 Discussion 2005), while limiting uptake of Mg, which is often excessively present in serpentine soils (Proctor, 1971; Brady et al., 2005). The pairs of plant species we studied with different degrees In our study, non-endemic serpentine plants maintained high- of serpentine habitat affinity differed in their acquisition and lo- er Ca concentrations in leaves than endemics despite having calization of nutrients and metals. Our results support the hy- lower leaf Mg concentrations. In fact, non-endemics had high- pothesis that serpentine endemics concentrate some nu- er Ca : Mg ratios across all tissues relative to endemics trients (i.e., K) into their tissues more than non-endemic spe- (Fig. 1). This pattern, contrary to our prediction, may be ex- cies. However, our results support the hypothesis that they plained by high Mg requirements, i.e., plants require higher will also exclude phytotoxic heavy metals to a greater degree external and internal Mg concentrations for optimal growth than non-endemics only for the metal Co, but not Ni or Cr, (reviewed in Brady et al., 2005), for serpentine endemics suggesting that there may be more common responses to (Palm et al., 2012). For example, Main (1981) found that Poa this aspect of serpentines soils. Endemic and non-endemic curtifolia (Poaceae), a serpentine endemic, required abnor- species clearly differed with respect to the uptake of several mally high concentrations of soil Mg to achieve optimal elements found either in excess or limiting supply in serpen- growth. Likewise, Madhok (1965) and Madhok and Walker tine soils; thus, our data lend support to the ‘specialist’ model (1969) found that the serpentine endemic Helianthus bolan- of serpentine endemism. deri ssp. exilis (Asteraceae) required higher internal concentrations of Mg relative to the related sunflower Helianthus an- If endemics are better adapted to the serpentine environment nuus, a non-serpentine species. Therefore, some serpentine than non-endemics, then we would expect serpentine endem- endemics may require higher internal Mg concentrations for ics to more effectively cope with low Ca concentrations rela- optimal growth, supporting the idea the endemics perform tive to non-endemics by selectively taking up Ca despite high better on serpentine compared to non-serpentine soils (spe- external Mg concentrations. Such a pattern has been found in cialist model), and this increased Mg uptake may correspond- comparisons of serpentine and non-serpentine congeners. ingly limit Ca uptake due to antagonistic effects (Brady et al., For example, O’Dell et al. (2006) found that serpentine spe- 2005; White, 2012). cies were able to maintain higher Ca concentrations, relative to Mg, in leaves compared to non-serpentine congeners In addition to Ca and Mg, endemics and non-endemics were when grown in serpentine soils. In this case, the plant species found to differ in K concentrations across all tissues (Fig. 1). adapted to serpentines were more efficient at acquiring Ca, These results corroborate the findings of Lee and Reeves which is generally limiting in serpentine soils (Brady et al., (1989), who found that the serpentine endemic Celmisia spe-

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0.0014 –1 centrations of P, Cr, and Ni among tissues. Furthermore, while 0.0012 Co endemics incorporated 56% less Co than non-endemics, con- 0.0010 centrations in both were generally low (mean for all tissues –1 0.0008 < 1 mg kg ; Fig. 2). While phytoavailable concentrations of Co and Cr were low at our sites, Ni concentrations were rela- 0.0006 tively high in comparison to reported phytoavailable Ni on 0.0004 non-serpentine soils (Berglund et al., 2003). Surveys of 0.0002 Concentrationkg / g phytoavailable element concentrations in other serpentine 0 soils have determined that excess Ni is the most important Leaves Flowers Seeds edaphic factor controlling serpentine vegetation (Robinson et al., 1996). Therefore, our results support the notion that resistance to elevated concentrations of soil Ni is critical for

–1 0.018 0.016 Cr growth on serpentine regardless of whether plants are en- 0.014 demic or non-endemic. Likewise, there was no effect of habi- 0.012 tat affinity on the uptake of P, which is a nutrient often limiting 0.010 in serpentine soils (Nagy and Proctor, 1997b) corroborating 0.008 the findings of O’Dell and Claussen (2006). These authors 0.006 found no significant differences between serpentine and non- 0.004 serpentine Achillea millefolium accessions with respect to Concentration / g kg g / Concentration 0.002 plant P concentrations when grown in P-limited serpentine 0 soils. Furthermore, phytoavailable P concentrations were low Leaves Flowers Seeds and within a range limiting on similar serpentine sites (Nagy and Proctor, 1997b). Our findings suggest that, while endem- 0.018 ics and non-endemics interact differently with some elements –1 Ni 0.016 of serpentine soils, plant persistence on serpentine requires 0.014 common physiological responses, such as the ability to ac- 0.012 quire key nutrients (e.g., P) while resisting elevated concen- 0.010 trations of potentially toxic Ni. 0.008 0.006 While our study describes patterns rather than processes, we 0.004 suggest that gene flow between serpentine and non-serpen- Concentrationkg / g 0.002 tine populations of non-endemic species may explain, at least 0 in part, the differences observed in our study between endem- Leaves Flowers Seeds ics and non-endemics. However, while extensive gene flow Figure 2: Mean Co, Cr, and Ni tissue concentrations of serpentine between serpentine and non-serpentine populations of non- endemic and non-endemic species. Dark bars indicate endemics, endemic species has been observed (e.g., Sambatti and while white bars indicate non-endemics. Error bars represent 95% Rice, 2006) and may limit their physiological efficiency (i.e., confidence intervals. nutrient uptake and metal exclusion) when on serpentine, local adaptation can still occur in some species (e.g., Gonzalo- Turpin and Hazard, 2009). Thus, further research is required denii (Asteraceae) was able to maintain higher internal con- to explain the differences observed and will continue to pro- centrations of K relative to a non-endemic congener, C. mar- vide insight into the nutritional requirements for plants grow- kii, when grown on serpentine soil. Potassium may be critical ing on serpentines. for plants on serpentines as it confers resistance to both biotic and abiotic stressors (Marschner and Cakmak, 1989; Gupta et al., 1989; Cakmak, 2005; Prabhu et al., 2007). However, it Acknowledgments must be acknowledged that the differences in K observed between endemic and non-endemic species may not be biologi- We thank C. Koehler and P. Aigner for field assistance, cally relevant as tissue K concentrations determined in both R. McKown for laboratory assistance, G. Arceo-Go´ mez for groups of plants are generally considered adequate for plant field and statistical assistance, and members of the Ashman growth and metabolism (reviewed in White, 2013). Further ex- lab for discussion. This research was funded by The Univer- perimental work would be required to determine if these differ- sity of Pittsburgh’s Office of Undergraduate Research, ences in K tissue concentrations would translate into differen- Scholarship, and Creative Activity,aBrackenridge Research ces in plant fitness of endemic and non-endemic serpentine Fellowship and a Howard Hughes Medical Institute Graduat- species. ing Senior Fellowship to K. S. DeHart, an Ivy McManus Di- versity Fellowship and an Andrew Mellon Predoctoral Fel- Despite differences in Ca, Mg, K, and Co, we found no signifi- lowship to G. A. Meindl, NSF (EAR-IF 0948366) to D. J. Bain, cant difference between endemics and non-endemics in con- and NSF (DEB 1020523) to T. L. Ashman.

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